Phosphoenol pyruvate (PEP) is a highly functionalized, chemically versatile molecule involved at several intersections of cellular energy metabolism and biosynthesis. While most enzymatic reactions utilizing PEP as a substrate involve cleavage of the high energy P-O, two types of reactions have been shown to involve the unusual cleavage of the C-O bond of PEP. These enzyme reactions fall in to two classes: (1) the first class is those that carry out an enolpyruvoyl transferase reaction and (2) the second class are those that carry out what is formally a net aldol condensation reaction. Representative enzymes in the enolpyruvoyl transferase class include EPSP synthase and MurZ. To date, there are only two known enzymes that carry out the net aldol condensation between PEP and the aldehyde moiety of a cosubstrate sugar: KDO8P synthase and DAHP synthase. Recently, a third enzyme, N-acetyl-neuraminic acid synthase, has been discovered that also appears to fall in to this category. Each of these bacterial enzymes is responsible for catalyzing the formation of key components such as lipopolysaccharide, aromatic amino acids, and capsular polysaccharides that are found in unique biosynthetic pathways and therefore may represent novel target enzymes for the design of new antibiotics. While the mechanistic aspects of the catalytic pathway for the enolpyruvoyl transferase have been delineated, the details of the catalytic mechanism for those enzymes in the second class that are involved in the net aldol condensation remain elusive. This proposal will investigate the catalytic mechanism for each of the three enzymes that catalyze a net aldol condensation and define the second class of enzyme that cleave the C-O bond of PEP. The two specific aims of this proposal are: 1) Elucidation of the KDO8P synthase molecular mechanism using transient kinetic methods to study: a) structure guided site-directed enzyme mutants and b) a series of alternate substrate and reaction intermediate analogs; and 2) Detection and characterization of novel enzyme intermediate from a series of three PEP-utilizing enzymes using a novel rapid mixing, pulsed-flow ESI-MS technique. These enzymes carry out a unique aldol type condensation of PEP and sugars of increasing length: C4 (DAHP synthase), C5 (KDO8P synthase), and C-6 (N-acetyl-neuraminic acid synthase). In addition as the investigator develops the quantitative aspects of the rapid mixing, pulsed-flow ESI-MS technique further, she will extend the studies of these enzymes to determine the full kinetic profile.